Rev Bras M ed Esporte _ Vol. 12, Nº 5 – Set/Out , 2006
233e
1. Universidade Estadual Paulista – UNESP. Departamento de EducaçãoFísica, Rio Claro / SP, Brasil.
2. Faculdades Integradas Einstein de Limeira – FIEL, Limeira/ SP, Brasil. 3. Faculdades Integradas de Bauru – FIB, Bauru / SP, Brasil.
Received in 15 / 9 / 05. Final version received in 19/ 12/ 05. Approved in 14 / 5 / 06. Correspondence to: Fúlvia de Barros M anchado, Av. 24A,1.515, Bela Vis-ta – 13506-900 – Rio Claro, SP – Brazil. Tel.: (19) 3527-0691, fax: (19) 3536-4974. E-mail: fbmanchado@yahoo.com.br
The maximal lactate steady state is ergometer-dependent
in experimental model using rats
Fúlvia de Barros M anchado1,2, Claudio Alexandre Gobatto1,
Ricardo Vinícius Ledesma Contarteze1, M arcelo Papoti1,3 and M aria Alice Rostom de M ello1
O
RIGINALA
RTICLEKeyw ords: Blood lactate. Sw imming. Treadmill running. Wistar rats.
ENGLISH VERSION
ABSTRACT
The maximal lactate steady state (M LSS) is considered the gold standard method for determination of aerobic /anaerobic metabo-lic transition during continuous exercise, but the blood lactate re-sponse at this intensity is ergometer-dependent in human beings. An important tool for exercise physiology and correlated fields is the use of animal models. How ever, investigation on evaluation protocols in rats is scarce. The aim of the present study w as to verify if the M LSS is ergometer-dependent for the evaluation of the aerobic conditioning of rats. Therefore, 40 adult male Wistar rats w ere evaluated in tw o different exercise types: sw imming and treadmill running. In both, the M LSS w as obtained w ith 4 conti-nuous 25 minutes tests, at different intensities, performed at 48 hours intervals. In all tests, blood samples w ere collected from a cut at the tail tip every 5 minutes for blood lactate analysis. The sw imming tests occurred in a deep cylindrical tank, w ith w ater temperature at 31 ± 1°C. The loads used in the test s w ere 4.5; 5.0; 5.5 and 6.0% of the body w eight tied to the animal’s back. For M LSS determination in running exercise, there w as selection of running rats and velocities used in the tests w ere 15, 20, 25, 30 m.min-1. The M LSS w as interpreted as an increase not exceeding 1.0 mM of blood lactate, from the 10th to the 25th minute of exer-cise. The M LSS in sw imming exercise occurred at 5.0% of body w eight (bw ), w ith blood lactate at 5.20 ± 0.22 mM . The running rats presented M LSS at the 20 m.min-1 velocity, w ith blood lactate of 3.87 ± 0.33 mM . The results indicated that the M LSS is ergom-eter-dependent in experimental models using animals, as it is in human beings.
INTRODUCTION
The determination of the metabolic predominance for the ener-gy supply during an exercise presents extreme importance for the correct prescription of an activity. Accordingly, there are many pro-posed evaluation protocols in order to detect the intensity of tran-sition betw een the aerobic and anaerobic metabolisms (Wasser-man and M cIloroy, 1964; M onod and Scherrer, 1965; Kinder(Wasser-man et al., 1979; Sjödin and Jacobs, 1981; Chassain, 1986 and Tegtbur et al., 1993).
Exercise physiology and correlated fields have been developing simple and complex methodologies over the years, w ith the pur-pose to determine the effort intensity. According to Gaesser and Poole (1996), the physiological responses facing the exercise trust-fully signal the characteristic of the predominant metabolism of the energy supply for the given activity. Among these responses it is possible to highlight the blood lactacidemia.
The maximal lactate steady state (M LSS), defined as the high-est intensity in w hich the anaerobic metabolism still predominates over the aerobic, is currently considered the gold standard method for the determination of the transition intensity betw een these metabolisms in continuous exercise performed by humans (Beneke, 1995; Beneke, 2003, Billat et al., 2003). According to Beneke et al. (1995), the lactacidemic response in this intensity is dependent on the ergometer used by these individuals, w hich implies in careful generalizations of mistaken information on the training loads pre-scribed by the concentration of blood lactate.
The importance to precisely determine the exercise intensity is not restricted to w orks in w hich humans are objects of the study. A tool that has been considered interesting to the organism obser-vation facing effort is the use of experimental models w ith ani-mals. Such models have been elaborated in order to simulate phys-iopathological or training-related situations occurred w ith humans, and solve occasional problems derived from the observed alter-ations (Oliveira et al., 2005; Braga et al., 2004; M urdes et al., 2004). With this aim, Gobatto et al. (2001) evaluated the M LSS in sed-entary rats adapted to the aquatic environment, submitted to the sw imming exercise, as proposed by Heck et al. (1985) for evalua-tion in humans. Thereby, the animals performed 20 minutes of continuous effort in intensities correspondent to 5, 6, 7, 8, 9 and 10% of their body w eight attached to their bodies, w ith blood sam-ples collection being conducted at every five minutes for later lac-tacidemia determination. The authors observed M LSS in intensi-ties correspondent to 6% of the body w eight, w ith stabilization concentration of 5,5 mM . This index is different and higher than the one reported for humans in distinct exercises and for rats per-forming effort in rolling treadmill (approximately 4 mM ). In a recent review on the maximal lactate steady state, Billat et al. (2003) point out the findings by Gobatto et al. (2001) as representative for the study w ith animals.
Other evaluation protocols have been developed in sw imming, such as the determination of the critical load and anaerobic w ork ability (M arangon et al., 2002) and minimal lactate test (Voltarelli et al., 2002). How ever, due to its great importance, all of them use the M SLL as gold Standard in order to validate their procedures.
234e
Rev Bras M ed Esporte _ Vol. 12, Nº 5 – Set/Out, 2006 similar to the one described for humans, w ith the sameconcentra-tion associated w ith the Lan, indeed. How ever, in the rats sw im-ming, Gobatto et al. (1991) did not find this classic exponential behavior of the blood lactate curve, w hich suggests the need of further investigation on the lactate kinetic in animals submitted to physical exercise and the lactate responses distinction in different ergometers.
The aim of the present study w as to evaluate w hether the max-imal lactate steady state is dependent on the ergometer used for the aerobic evaluation in rats, as it is for humans, due to this meth-od’s importance and also for prevention of mistakes in the pre-scription of activity for animals in different kinds of exercise.
M ATERIALS AND M ETHODS
Animals
All experiments w ere conducted according to the American College of Sports M edicine politics and approved by the Biosciences Institute of the São Paulo State University – UNESP, Rio Claro. Forty-four Wistar rats, w ith 90 days of age, w eighting 443 ± 33 g, w ere used. During the experimental period, the animals w ere kept in collective cages (five rats per cage) in a lighted room w ith light-dark cycle of 12:00-12:00 hs and temperature of 25°C. The rats received commercial food specific for rodents (Labina-Purina) and w ater ad libitum.
Experimental protocol
Adaptation to the w ater environment
Tw enty animals w ere submitted to maximal lactate steady state tests in sw imming exercise. The rats w ere adapted to the w ater environment in a standardized manner prior to the tests conduc-tion. The adaptation occurred in a total period of 15 uninterrupted days, in a cylindrical tank w ith smooth surface, measuring 60 cm of diameter by 120 cm of depth (M arangon et al., 2001), w ith w a-ter temperature kept at 31 ± 1°C (Harri and Kuusela , 1986). The purpose of the adaptation w as to decrease the animal’s stress w ith-out promoting physiological adaptations derived from the physical training, though.
Initially, the rats w ere inserted in shallow w ater for three days during 15 minutes. Later, the w ater level w as increased, as w ell as the effort duration time and the load to be held by the animal. Thus, on the forth day, the rats sw am in deep w ater for tw o minutes, w ith increase of tw o minutes at each day until the tenth day of adaptation. On the eleventh day, the animals w ere submitted to the sw imming exercise for five minutes holding a load of 3% of their body w eight, w ith increase of five minutes at each day, w hen, on the fifteenth day, the adaptation ceased.
Selection of the runner rats and adaptation to the treadmill
It w as necessary to previously select the runner animals in or-der to conduct the tests in treadmill. The selection occurred in a seven day-period, in w hich the 20 rats that presented positive re-sponse to the running stimulus at least five times w ere chosen After the selection, the animals w ere submitted to an adaptation to the treadmill exercise, w ith progressive velocities (5 to 20 m.min -1) and duration (5 to 15 min). The aim of the adaptation w as also the reduction of stress indices presented by the animal due to the task being know n w ithout physical training promotion.
Determination of the maximal lactate steady state
The w hole experimental protocol w as conducted in environmen-tal conditions identical to the ones during the adaptation period, both in sw imming and in treadmill running.
In both exercises, the protocol for the M LSS determination con-sisted of five continuous tests w ith duration of 25 minutes, in dif-ferent effort intensities, randomly distributed and separated by a
48-hour resting interval. In all tests there w as blood collection of the animals‘ tails in the resting times, 5, 10, 15, 20 and 25 minutes of exercise, for later blood lactate analysis and the lactacidemic curves in each intensity.
Sw imming tests
The rats performed 25 minutes of continuous effort in loads equivalent to 4,5; 5,0; 5,5 and 6,0% of their body w eight, attached to their backs. There w as daily load adjustment, w ith the animals‘ body mass measurement. Blood samples from the animals‘ tails w ere performed for each intensity in the times previously described, and the blood sample’s treatment occurred according to a tech-nique detailed below.
Treadmill running tests
For the M LSS evaluation in running, the selected animals per-formed the continuous tests in the 15, 20, 25 and 30 m.min-1 ve-locities. The treadmill specific for training w ith rats composed of eight lanes, w as kept w ith the electric shock off, reducing hence, the stress effect in the effort conducted by the animal. Blood sam-ples w ere removed from the rats‘ tails as in sw imming.
Blood samples and analysis
During the continuous tests, blood samples (25 µl) w ere removed from the animal’s tail in the times already described and placed in Eppendorf tubes (capacity of 1,5 ml), containing 50 µl of sodium fluoride (1% ). In order to avoid the blood dilution in w ater in the case of the sw immers, the animals w ere removed from the cylin-der and dried, returning to the aquatic environment immediately after the blood collection. The blood lactate concentrations w ere determined in a lactate analyzer (M odel YSI 1500 Sport, Yellow Springs, OH, EUA).
Statistical analysis
In both ergometers the M LSS w as interpreted as the highest exercise intensity in w hich the increase of the lactacidemia w as equal or low er than 1 mM , from the 10th to the 25th minute. The average concentrations of blood lactate in each intensity for the tw o ergometers w ere obtained through the ratio of the lactaci-demic indices of the times 10, 15, 20 and 25 minutes of exercise. A one-w ay ANOVA w as used in order to identify the differences betw een the blood lactate concentrations in the several durations of the continuous exercise and betw een distinct ergometers: sw im-ming and treadmill running. The results are expressed in average ± standard error of the average. In all statistical procedures, the significance index w as preset in P < 0,05 (Daw son-Saunders and Trapp, 1994).
RESULTS
The blood lactate curves of the rats submitted to the sw imming exercise are expressed in figure 1. The M LSS w as observed in the intensity equivalent to 5% of the body w eight, in an average lac-tate concentration of 5,20 ± 0,22 mM .
In the running exercise, the velocity correspondent to the max-imal lactate steady state w as 20 m.min-1, in lactate concentration of 3,87 ± 0,33 mM , though (figure 1). In the intensity equivalent to 30 m.min-1 only 20% of the animals completed the test.
The one-w ay ANOVA identified significant difference betw een the maximal lactate steady state obtained in the sw imming exer-cise and the treadmill running.
DISCUSSION
Rev Bras M ed Esporte _ Vol. 12, Nº 5 – Set/Out , 2006
235e
ificity of the w ater tank used. In the present w ork, it w as chosen to perform the sw imming exercise in a deep tank (60 cm of diameter by 120 cm of depth) w ith a smooth surface, avoiding thus, that the animals could lean on the sides of the tank during the test or could touch the bottom of the tank performing a jump movement. In the study by Gobatto et al. (2001), the tanks used w ere not deep (100 cm of diameter by 80 cm of depth), w hich possibly favored the rats‘ activity.The stabilization concentration of the lactate in the maximal aer-obic intensity in sw imming w as 5,20 ± 0,22 mM , an index very close to the one described by Gobatto et al. (2001) for sedentary rats (5,5 mM ), w hich suggests reproducibility of this concentration for the species of evaluated animals. Even after aerobic training, Gobatto et al. (2001) did not observe alteration in the lactate stabi-lization concentration. In humans, the lactate stabistabi-lization in maxi-mal aerobic intensity is usually found betw een 3 and 7 mM (Steg-mann et al., 1981; Harnish et al., 2000), how ever, in sw imming exercise, this index seems to be close to the low er threshold of the described group. Pereira et al. (2002) determined the anaero-bic threshold of sw imming athletes in progressive test and ob-served inflexion of the lactacidemic curve in 3,5 mM concentra-t ion. Thus, iconcentra-t seem s concentra-t haconcentra-t W isconcentra-t ar raconcentra-t s presenconcentra-t higher lacconcentra-t aconcentra-t e concentration indices in the M LSS in sw imming exercise w hen compared to humans.
In the treadmill running w e obtained M LSS in 20 m.min-1 intensi-ty in 3,87 ± 0,33 mM concentration. Pillis et al. (1993) and Langfort et al. (1996) suggest the occurrence of the anaerobic threshold in runner rats in the 25 m.min-1 velocity, w hich is higher than in our findings. In the present study, the animals w ere submitted to 25 minutes of continuous exercise in the 15, 20, 25 and 30 m.min-1 velocities, allow ing a longer observation of the blood lactate kinet-ic, w hile those authors only observed the lactate behavior related to progressive exercise.
Concerning the lactate concentration, the treadmill exercise pro-moted stabilization in an index significantly low er to the one ob-served in sw imming (figures 1 and 2). The lactacidemic concentra-tion verified in the present study is similar to the point of inflexion of the lactate curve described by Pillis et al. (1993) (4 mM ) and to the index obtained in a classic study performed w ith humans in this kind of exercise (Heck et al., 1985). There are no w orks in the literature w hich present aerobic training results in running in max-imal lactate steady state w ith Wistar rats, as the one conducted by Gobatto et al. (2001) in sw imming.
Our results clearly demonstrate the ergometer dependence of the maximal lactate steady state protocol in exercises for Wistar rats, as w ell as in humans. Such fact demands caution in the eval-uation and prescription of physical training for these animals. Fur-ther studies should be conducted w ith rats in different physiopatho-logical and training conditions in order to identify possible alterations in the evaluated aerobic parameter.
ACKNOWLEDGM ENTS
This study w as supported by the Foundation of Research Support of the São Paulo State (FAPESP – Proc. 07070-5/2004) and the Research Centers CAPES and CNPq (Proc. 300270/2004-16). We also thank José Roberto Rodrigues da Silva, Eduardo Custódio and Clarice Yoshiko Sibuya for the technical support.
All the authors declared there is not any potential conflict of inter-ests regarding this article.
REFERENCES
1. Wasserman K, M cIlroy M B. Detecting the threshold of anaerobic metabolism in cardiac patients during exercise. Am J Cardiol 1964;14:844.
2. M onod H, Scherrer J. The w ork capacity of a synergic muscular group. Ergo-nomics 1965;8:329-38.
a
b
Figure 1 – M aximal blood lactate steady state obtained in sw imming exer-cise for rats (a) (n = 20) and treadmill running (n = 20) (b). The results are expressed in average ± average standard error. The lactacidemia during the exercise w as higher than the initial in all cases. * significant difference betw een the lactacidemia at 5 minutes and the remaining blood lactate indices in all exercise intensities. # significant difference betw een the blood lactate indices in the 6% intensity of the remaining intensities, from the tenth to the tw enty-fifth minute of exercise § significant difference be-tw een the lactacidemia from the fifteenth to the be-tw enty-fifth minute and the lactate indices at 5 minutes, in the velocities equivalent to 25 and 30 m.min-1. x significant difference betw een the lactacidemic curves in the 25 and 30 m.min-1 velocities compared to the lactate curves obtained at 15 and 20 m.min-1, from the fifteenth to the tw enty-fifth minute of exercise.
effort. Such fact implies in the use of this parameter as a prescrip-tion instrument (M ujika et al., 1995) and sports training aid (Pyne et al., 2001).
The maximal lactate steady state is related to the biochemical balance betw een appearance and removal of the blood lactate (M ader and Heck, 1986) and is considered the highest exercise intensity in w hich this steady state still occurs, outlining hence, a transition of aerobic-anaerobic metabolic predominance (Beneke, 1995; Billat et al., 2003). Therefore, it is considered a safe method for aerobic ability identification (Jones and Carter, 2000), w hich corroborates so that many studies use this procedure in the evalu-ation of active individuals and high performance athletes (Jones and Doust, 1998; Harnish et al., 2001; Beneke, 2003).
Our study is important due to the application of this individual protocol in order to evaluate laboratory animals in tw o different kinds of exercise: sw imming and treadmill running. M oreover, there is massive scientific meaning in obtaining data about probable dif-ferences of lactacidemic responses in tw o ergometers w idely used for training of Wistar rats.
spec-236e
Rev Bras M ed Esporte _ Vol. 12, Nº 5 – Set/Out, 20063. Kindermann W, Simon G, Keul J. The significance of the aerobic-anaerobic tran-sition for the determination of w ork load intensities during endurance training. Eur J Appl Physiol 1979;42:25-34.
4. Sjödin B, Jacobs I. Onset of blood lactate accumulation and marathon running performance. Int J Sports M ed 1981;2:23-6.
5. Chassain A. M éthode d’appréciation objetive de la tolérance de l’organisme à l’effort: application à la mensure des puissances de la frequence cardiaque et de la lactatémie. Science & Sports 1986;1:41-8.
6. Tegtbur U, Busse M W, Braumann KM . Estimation of an individual equilibrium betw een lactate production and catabolism during exercise. M ed Sci Sports Ex-erc 1993;25:620-7.
7. Gaesser GA, Poole DC. The slow component of oxygen uptake kinetics in hu-mans. Exerc Sports Sci Rev 1996;24:35-70.
8. Beneke R. Anaerobic threshold, individual anaerobic threshold, and maximal lac-tate steady slac-tate in row ing. M ed Sci Sports Exerc 1995;27:863-7.
9. Beneke R. M ethodological aspects of maximal lactate steady state-implications for performance testing. Eur J Appl Physiol 2003;89:95-9.
10. Billat VL, Siverent P, Py G, Korallsztein J-P, M ercier J. The concept of maximal lactate steady state: a bridge betw een biochemistry, physiology and sport sci-ence. Sports M ed 2003;33:407-26.
11. Oliveira CAM , Luciano E, M ello M AR. The role of exercise on long-term effects of alloxan administered in neonatal rats. Experimental Physiology 2005;90:79-86.
12. Braga LR, M ello M AR, Gobatto CA. Exercício contínuo e intermitente: efeitos do treinamento e do destreinamento sobre a gordura corporal de ratos obesos. Archivos Latinoamericanos de Nutrición 2004;54:58-65.
13. M urdes JP, Bortel R, Blanknhin EP, Rossin AA, Garnier DL. Rats models of type 1 diabetes: genetics, environment and autoimmunity. Ilac J 2004;45:278-91. 14. Gobatto CA, M ello M AR, Sibuya CY, Azevedo JRM , Santos, LA, Kokubun E.
M aximal lactate steady state in rats submitted to sw imming exercise. Comp Biochem Physiol 2001;130A:21-7.
15. Heck H, M ader A, Hess G, M ücke S, M üller R, Hollmann W. Justification of the 4-mmol/L lactate threshold. Int J Sports M ed 1985;6:117-30.
16. M arangon L, Gobatto CA, M ello M AR, Kokubun E. Utilization of an hiperbolic model for the determination of the critical load in sw imming rats. M ed Sci Sports Exerc (Suppl) 2002;34:149.
17. Voltarelli FA, Gobatto CA, M ello M AR. Determination of anaerobic threshold in rats using the lactate minimum test. Braz J M ed Biol Res 2002;35:1-6. 18. Pillis W, Zarzeczny R, Langfort J, Kaciuba-Uscilko H, Nazar K, Wojtyna J.
Anaer-obic threshold in rats. Comp Biochem Physiol 1993;106A:285-9.
19. Gobatto CA, Kokubun E, Sibuya CY, M ello M AR. Efeitos da desnutrição protéi-co-calórica e do treinamento físico na produção de ácido lático em ratos machos adultos após teste de cargas progressivas. Resultados preliminares. Ciência e Cultura 1991;43:725-6.
20. Harri M , Kuusela P. Is sw imming exercise or cold expose for rats? Acta Physiol Scand 1986;126:189-97.
21. Daw son-Saunders B, Trapp RG. Basic and clinical biostatistic. East Norw alk, Con-necticut: Appleton and Lange, 1994.
22. M ujika I, Chatard JC, Busso T, Geyssant A, Barale F, Lacoste L. Effects of train-ing on performance in competitive sw immtrain-ing. Can J Appl Physiol 1995;20:395-406.
23. Pyne B, Lee H, Sw anw ick KM . M onitoring the lactate threshold in w orld ranked sw immers. M ed Sci Sports Exerc 2001;33:291-7.
24. M ader A, Heck H. A theory of metabolic origin of the anaerobic threshold. Int J Sports M ed 1986;7:45-65.
25. Jones AM , Carter H. The effect of endurance training on parameters of aerobic fitness. Sports M ed 2000;29:373-6.
26. Jones AM , Dousty JH. The validity of the lactate minimum test for determina-tion of the maximal lactate steady state and physiological correlates to 8 km running performance. M ed Sci Sports Exerc 1998;30:1304-13.
27. Harnish CR, Sw ensen TC, Pate RP. M ethods for estimating the maximal lactate steady state in trained cyclists. M ed Sci Sports Exerc 2001;33:1052-5. 28. Stegmann H, Kindermann W, Schanabel A. Lactate kinetics and individual
anaer-obic threshold. Int J Sports Exerc 1981;2:160-5.
29. Pereira RR, Zagatto AM , Papoti M , Gobatto CA. Validação de dois protocolos de teste para determinação do limiar anaeróbio em natação. M otriz 2002;8:63-8. 30. Langfort J, Zarzeczny R, Pillis W, Kaciuba-Uscilko H, Nazar K, Porta S. Effect of
